Atherosclerosis is the primary cause of heart attack, stroke, and peripheral artery disease, which collectively account for >30% of all deaths in the US. Multiple studies recognized abnormal cholesterol homeostasis as a risk factor for the development of atherosclerosis. We and others have recently reported on miR-33, a conserved microRNA that is encoded within intron 16 of SREBP-2. The clinical importance of SREBP-2 is revealed in hypercholesterolemic patients treated with statins, which reduce LDL-cholesterol levels by increasing hepatic expression of SREBP-2 and its target the LDL-receptor. Our published and preliminary studies show that physiological targets of miR-33 include ABCA1, a transporter critical for HDL lipidation and reverse cholesterol transport, and ATP8B1, a phospholipid flippase linked to intrahepatic cholestasis. The broad goal of this proposal is to determine the role of miR-33 on cholesterol, bile and lipoprotein homeostasis. We hypothesize that miR-33 controls key aspects of sterol mobilization, bile excretion and HDL metabolism. Importantly, since statins are usually prescribed to hypercholesterolemic patients, and statins induce the expression of miR-33, we speculate that statin-treated patients will have elevated levels of miR-33 and persistent down regulation of ABCA1 and ATP8B1 by miR-33. To address these questions, we propose the following 3 specific aims: 1) Determine the role of miR-33 in hepatic sterol homeostasis in primary hepatocytes and in mice, by measuring the effect of over expression or silencing of miR-33 on lipoprotein secretion and bile excretion; 2) Test the hypothesis that altered macrophage miR-33 expression results in changes in reverse cholesterol transport and atherosclerotic lesion size, by performing experiments in vivo to determine the role of macrophage miR-33 expression on cholesterol -/- mobilization and progression of atherosclerotic lesions in Ldlr mice ; and 3) Test the hypothesis that systemic silencing of miR-33 is atheroprotective in vivo, by characterizing the composition and biological properties of HDL generated following systemic silencing of miR-33, and by evaluating whether anti-miR-33 oligonucleotides synergize with the atheroprotective effects of statins in ApoE*3Leiden W hCETP mice. There are still multiple aspects of cholesterol homeostasis, cholestasis and atherogenesis that remain obscure. Our data suggest that the cholesterol-miR-33 axis modulates key aspects of hepatocyte and macrophage biology. We anticipate that our studies will provide new clues into the complex regulatory networks that control intracellular cholesterol levels, bile excretion and circulating lipoproteins. If our hypothesis is true, miR-33 might be a target for novel therapies to manage dyslipidemias and/or cholestasis. !